# US 2019/0170762 A1 · Method for measuring glycated albumin

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- Number: US 2019/0170762 A1
- Document ID: US20190170762A1
- Status: Published application
- Kind: A1
- Application number: 16/320,814
- Filed: 2017-07-19
- Published: 2019-06-06
- Earliest claimed: 2016-07-28
- Assignees: Asahi Kasei Pharma Corporation
- Applicants: Asahi Kasei Pharma Corporation
- Inventors: Ryosuke KOGUMA
- Web page: https://patentgrep.com/patent/US20190170762A1

## CPC

- C12Q 1/26: involving oxidoreductase
- G01N 33/6827: {Total protein determination, e.g. albumin in urine}
- C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters
- G06F 17/11: for solving equations {, e.g. nonlinear equations, general mathematical optimization problems (optimization specially adapted for a specific administrative, business or logistic context G06Q10/04)}
- G01N 21/77: by observing the effect on a chemical indicator
- G01N 2333/765: Serum albumin, e.g. HSA
- G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation
- G16B 5/00: ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
- G16B 50/00: ICT programming tools or database systems specially adapted for bioinformatics
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G06F: ELECTRIC DIGITAL DATA PROCESSING (computer systems based on specific computational models G06N)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

An object of the present invention is to provide a method of measuring a glycated-albumin (GA) value traceable to a certified GA value conveniently at a low cost in a short period of time. A method of measuring a GA value traceable to the certified GA value includes the following steps: A1) determining Regression equation III by steps including the following b), c), and e0) to h); b) determining a GA concentration [L] (A) of two or more GA certified reference materials having respectively different certified GA values; c) measuring a GA-derived absorbance (D) of the two or more GA certified reference materials; e0) creating Linear equation II0 having an intercept of zero; f) determining two or more GA concentrations[P] (E) proportionate to the GA-derived absorbance (D) according to Equation II0; g) determining two or more GA values (F); and h) creating Regression equation III.

## Claims

### Claim 1

A method of measuring a glycated-albumin (GA) value traceable to a certified GA value, the method comprising:
A0) determining a regression equation for converting:
a measured value of a GA value of a sample material into a certified GA value of a GA certified reference material;
a measured value of a GA concentration[P] of the sample material into a GA concentration[L] of the GA certified reference material;
the measured value of the GA value of the sample material into a GA value set for the sample material from the certified GA value of the GA certified reference material; or
the measured value of the GA concentration[P] of the sample material into a GA concentration[L] set for the sample material from the GA concentration[L] of the GA certified reference material; and
F0) measuring a GA concentration[P] and an albumin concentration of the sample material by using a calibration curve prepared by using a reference material for which “a GA concentration[P] converted from the GA concentration[L] of the GA certified reference material” and “an albumin concentration converted from an albumin concentration of the GA certified reference material” are set and determining a GA value traceable to the certified GA value according to the regression equation determined in A0),
wherein, the GA concentration[L] in each of A0) and F0) is a concentration of glycated lysine residues (DOF-Lys) and the GA concentration[P] is a concentration of a glycated amino acid or glycated peptide.

### Claim 2

A method of measuring a glycated-albumin (GA) value traceable to a certified GA value, the method comprising:
A1) determining Regression equation III by which determination comprises the following b), c), and e0) to h):
b) determining respective GA concentrations[L] (A) of two or more GA certified reference materials having respectively different certified GA values according to the following Equation 1:
GA concentration[L] (A)=(certified GA value (B) of GA certified reference material)×(albumin concentration (C) of GA certified reference material) (Equation 1)
c) measuring respective GA-derived absorbances (D) of the two or more GA certified reference materials;
e0) creating the following linear equation II0 having an intercept of zero:
D=α0E (Equation II0);
wherein,
α0 is an arbitrary positive value and
E represents a GA concentration[P] (E) proportionate to the GA-derived absorbance (D)),
f) determining two or more GA concentrations[P] (E) proportionate to the GA-derived absorbances (D) according to the Equation II0;
g) determining two or more GA values (F) according to the following Equation 2:
GA value (F)=(GA concentration[P] (E))/(albumin concentration (C)) (Equation 2); and
h) creating the following Regression equation III from the respective certified GA values (B) of the two or more GA certified reference materials and the two or more GA values (F) corresponding thereto:
B=γF+δ (Regression equation III),
wherein the GA concentration[L] in each of A1), b), c), and e0) to h) is a concentration of glycated lysine residues (DOF-Lys) and the GA concentration[P] is a concentration of a glycated amino acid or a glycated peptide.

### Claim 3 (depends on 2)

The method according to claim 2, further comprising:
B) measuring a GA-derived absorbance (G) of a reference material and determining a GA concentration[P] (H) corresponding thereto according to the Equation II0, and
C) measuring an albumin-derived absorbance (I) of the reference material and determining an albumin concentration (J) of the reference material.

### Claim 4 (depends on 3)

The method according to claim 3, wherein C) includes the following C1) to C3):
C1) determining e that satisfies the following Equation 3 from an albumin-derived absorbance (K) of the GA certified reference material and the albumin concentration (C) of the GA certified reference material:
K=εC (Equation 3);
C2) measuring the albumin-derived absorbance (I) of the reference material; and
C3) determining the albumin concentration (J) of the reference material from the absorbance (I) according to the following Equation 3′:
I=εJ (Equation 3′).

### Claim 5 (depends on 3)

The method according to claim 3, further comprising, prior to b), the following a1) to a3):
a1) determining a molar certified albumin value (L) according to the following Equation 4:
molar certified albumin value (L)=(certified albumin value of albumin certified reference material)/(molecular weight of albumin) (Equation 4);
a2) measuring an albumin-derived absorbance (M) of the albumin certified reference material; and
a3) determining θ that satisfies the following Equation 5 from the molar certified albumin value (L) and the absorbance (M):
M=θL (Equation 5); and
(C) includes the following C2) and C4):
C2) measuring the albumin-derived absorbance (I) of the reference material; and
C4) determining the albumin concentration (J) of the reference material from the absorbance (I) according to the following Equation 5′:
I=θJ (Equation 5′).

### Claim 6 (depends on 2, 3, 4, 5)

The method according to any one of claims 2 to 5, further comprising the following F1) to F4):
F1) determining, from the GA-derived absorbance (D) of the GA certified reference material, a GA concentration[P] (N) corresponding thereto by using a calibration curve prepared by using the reference material;
F2) determining, from the albumin-derived absorbance (K) of the GA certified reference material used in F1), an albumin concentration (O) corresponding thereto by using a calibration curve prepared by using the reference material;
F3) determining a GA value (P) according to the following Equation 6:
GA value (P) of GA certified reference material=(GA concentration[P] (N))/(albumin concentration (O)) (Equation 6); and
F4) determining a GA value (Q) traceable to the certified value of the GA certified reference material used in F1) while using the GA value (P) and the Regression equation III.

### Claim at position 7

7-9. (canceled)

### Claim 10

A method of measuring a glycated-albumin (GA) value traceable to the certified GA value, the method comprising:
A3) determining Regression equation V by which determination comprises the following b) to e0) and j) to o):
b) determining respective GA concentrations[L] (A) of two or more GA certified reference materials having respectively different certified GA values according to the following Equation 1:
GA concentration[L] (A)=(certified GA value (B) of GA certified reference material)×(albumin concentration (C) of GA certified reference material) (Equation 1);
c) measuring respective GA-derived absorbances (D) of the two or more GA certified reference materials;
d) creating, from the two or more GA-derived absorbances (D) and the two or more GA concentrations[L] (A), the following Regression equation I:
D=αA+β (Regression equation I);
e0) creating the following Linear equation II0 having an intercept of zero:
D=α0E (Equation II0)
(wherein,
α0 is an arbitrary positive value and
E represents a GA concentration[P] (E) proportionate to the GA-derived absorbance (D)),
j) measuring respective GA-derived absorbances (S) of two or more sample materials and determining GA concentrations[L] J (T) corresponding thereto according to the Regression equation I;
k) measuring respective albumin-derived absorbances (U) of the two or more sample materials and determining albumin concentrations (V):
l) determining, from the GA concentrations[L] (T), respective GA values1 (W) of the two or more sample materials according to the following Equation 8:
GA value1 (W)=(GA concentration[L] (T))/(albumin concentration (V)) (Equation 8);
m) determining, from the respective GA-derived absorbances (S) of the two or more sample materials, GA concentrations[P] (X) corresponding thereto according to the Equation II0;
n) determining, from the GA concentrations[P] (X), respective GA values2 (Y) of the two or more sample materials according to the following Equation 9:
GA value2 (Y)=(GA concentration[P] (X))/(albumin concentration (V)) (Equation 9); and
o) creating, from the GA values1 (W) of the two or more sample materials and the two or more GA values2 (Y) corresponding thereto, the following Regression equation V:
W=πY+φ (Regression equation V),
wherein the GA concentration[L] in each of A3), b) to e0) and i) to g) is a concentration of glycated lysine residues (DOF-Lys) and the GA concentration[P] is a concentration of a glycated amino acid or a glycated peptide.

### Claim 11 (depends on 10)

The method according to claim 10, further comprising:
B) measuring a GA-derived absorbance (G) of a reference material and determining a GA concentration[P] (H) corresponding thereto according to the Equation II0; and
C) measuring an albumin-derived absorbance (I) of the reference material and determining an albumin concentration (J) of the reference material.

### Claim 12 (depends on 10, 11)

The method according to claim 10 or 11, further comprising:
F1′) determining, from the GA-derived absorbance (S) of the sample material, a GA concentration[P] (N′) corresponding thereto by using a calibration curve prepared by using the reference material;
F2′) determining, from the albumin-derived absorbance (U) of the sample material used in F1′), an albumin concentration (O′) corresponding thereto by using a calibration curve prepared by using the reference material;
F3′) determining a GA value (P′) according to the following Equation 6′:
GA value (P′) of sample material=(GA concentration[P] (N′))/(albumin concentration (O′)) (Equation 6′); and
F4′) determining, by using the GA value (P′) and the Regression equation V, a GA value (Q2′) traceable to a certified GA value1 (W) with which the sample material used in Step F1′) is assigned.

### Claim at position 11

13-15. (canceled)

### Claim 16 (depends on 1, 2, 3, 4, 5, 10, 11)

The method according to any one of claims 1 to 5, 10 and 11, wherein three or more GA certified reference materials are used.

### Claim 17 (depends on 1, 2, 3, 4, 5, 10, 11)

The method according to any one of claims 1 to 5, 10 and 11, wherein the certified GA value is a certified GA value determined by isotope dilution mass spectrometry (ID-MS).

### Claim 18 (depends on 1, 2, 3, 4, 5, 10, 11)

The method according to any one of claims 1 to 5, 10 and 11, wherein the certified GA value is a certified GA value determined by isotope dilution mass spectrometry (ID-MS) and the GA certified reference material is JCCRM 611.

### Claim 19 (depends on 2, 10)

A GA value measurement kit, comprising:
the Regression equation III determined by the method as claimed in claim 2 or the Regression equation V determined by the method as claimed in claim 10;
a GA measurement reagent;
an albumin measurement reagent; and
a reference material.